Phosphatidylalkanolamine derivatives

ABSTRACT

THERE ARE DISCLOSED HEREIN PHOSPHATIDYLALKANOLAMINES IN WHICH THE ALKANOLAMINE IS 2-HYDROXYETHYL-, 2- OR 3-HYDROXYPROPYL-, OR 3-HYDROXYBUTYLAMINE AND THE ACYL GROUPS CONTAIN FROM 18-20 CARBON ATOMS AND THREE OR MORE DOUBLE BONDS. THE COMPOUNDS HAVE ANTI-HYPERTENSIVE PROPERTIES, AND METHODS FOR THEIR PREPARATION AND USE ARE ALSO DISCLOSED.

United States Patent 3,577,446 PHOSPHATIDYLALKANOLAMINE DERIVATIVESSumanas Rakhit, Dollard des Ormeaux, Quebec, Canada, assignor toAmerican Home Products Corporation, New York, N.Y. No Drawing. FiledSept. 9, 1968, Ser. No. 758,587 Int. Cl. A23j 7/00 US. Cl. 260-403 1Claim ABSTRACT OF THE DISCLOSURE There are disclosed hereinphosphatidylalkanolamines in which the alkanolamine is 2-hydroxyethyl-,2- or 3-hydroxypropyl-, or 3-hydroxybutylamine and the acyl groupscontain from 18-20 carbon atoms and three or more double bonds. Thecompounds have anti-hypertensive properties, and methods for theirpreparation and use are also disclosed.

This invention relates to new phosphatidylalkanolamine derivatives andto processes used for their synthesis.

More specifically, this invention relates to phosphatidylalkanolaminederivatives of Formula I,

in which R and R represent the same or different acyl group containing18 to 20 carbon atoms and three or more double bonds, such as, forexample, an octadeca- 6,9,12-trienoyl, octadeca-9,12,15-trienoyl,octadeca-6,9, 12,15-tetraenoyl, eic0sa-8,11,14-trienoyl or aneicosa-5,8, 11,14-tetraenoyl group; n represents the integers one ortwo; and R represents a hydrogen atom or a lower alkyl group, such as,for example, a methyl group.

The phosphatidylalkanolamine derivatives of this invention have beenfound to possess pharmacological properties which render them useful asmedicinal agents. More particularly, these derivatives exhibit utilityas antihypertensive agents when tested in standard pharmacologicaltests. For example, when these derivatives are administered to renalhypertensive rats, obtained by the method of A. Grollman, Proc. Soc.Exptl. Biol. Med., 57, 102 (1944), reduction of blood pressure towardnormal levels is observed. This fall in blood pressure is readilymeasured by the method of H. Kersten et al., J. Lab. Clin. Med., 32,1090 (1947).

When the compounds of this invention are employed as antihypertensiveagents in warm-blooded animals, e.g. rats, alone or in combination withpharmacologically acceptable carriers, the proportion of which isdetermined by the solubility and chemical nature of the compound, chosenroute of administration and standard biological practice. For example,they may be administered orally in solid form containing such excipientsas starch, milk sugar, certain types of clay and so forth. They may alsobe administered orally in the form of solutions or they may be injectedparenterally. For parenteral administration they may be used in the formof a sterile solution containing other solutes, for example, enoughsaline or glucose to make the solution isotonic.

The dosage of the present therapeutic agents will vary with the form ofadministration and the particular compound chosen. Furthermore, it willvary with the particular host under treatment. Generally, treatment isinitiated with small dosages substantially less than the optimum dose ofthe compound. Thereafter, the dosage is increased by small incrementsuntil the optimum effect under the circumstances is reached. In general,the compounds of this invention are most desirably administered at aconcentration level that will generally afford efiective results withoutcausing any harmful or deleterious side eifects and preferably at alevel that is in a range of from about 0.3 mg. to about mg. per kilo perday, although as aforementioned variations will occur. However, a dosagelevel that is in the range of from about 3 mg. to about 10 mg. per kiloper day is most satisfactory. Such doses may be administered once ortwice a day, as required.

A noteworthy aspect of this invention is the discovery that a minimum ofthree double bonds must be present in the acyl groups of thephosphatidylalkanolarnines of Formula I before said compounds exhibitantihypertensive activity. For example,1,2-di-(octadeca-9,12-dienoyl)-snglycero 3 phosphorylethanolamine(containing two double bonds per acyl group) (I; R and R -CH (CH 3 (CHCH CH) 2 (CH COO CHzO HBO CH3 CHO on O H R III.

OH R.

in which R R R and n are as defined above and Z represents a primaryamino group coupled with an amineprotecting group, such as, preferably aphthalyl group or some other amine-protecting group, for example, suchas those described E. Schriider and K. Liibke in The Peptides, vol. I,Academic Press, New York, 1965, pp. 3-51.

The starting material of Formula H may be1,2-isopropylidene-sn-glycerol, described by E. Baer, Biochem. Prep, 2,31 (1952) or rac-1,2-isopropylidineglycero1, de-

3 scribed by M. S. Newman and M. Renoll, J. Am. Chem. Soc., 67, 1621(1945).

The starting material of Formula II is condensed with phosphorusoxychloride in the presence of an organic base, preferably quinoline,followed by treatment with the appropriate 2- or3-hydroxyalkylphthalimide described below. In this manner theglycerophosphoric acid diester acetonide of Formula III is obtained.

The 2- and 3-hydroxyalkylphthalimides preferred in the above reactionare N-(2-hydroxyethyl)-, N-(3-hydroxypropyl)-, N-(2-hydroxypropyl)- andN-(3-hydroxybutyl) phthalimides. The first two compounds are describedby F. Garelli and G. Racciu, Atti accad. sci. Torino, Classe sci. fis.,mat. nat., 69, 358 (1934); Chem. Abstn, 29, 6223 (1935); the latter twocompounds are described by S. Gabriel and H. Ohle, Chem. Ber., 50, 819(1917) and R. Robinson and H. Suginome, J. Chem. Soc., 304 (1932),respectively.

The glycerophosphoric acid diester acetonide of Formula III is subjectedto mild hydrolyzing conditions, preferably by bringing said diester ofFormula III into contact with an acidic ion exchange resin in thepresence of an aqueous medium, such as 80% aqueous methanol. Suchconditions remove the acetone group and yield the correspondingglycerophosphoric acid diester of Formula Alternatively, hydrolysis witha dilute acid, such as, for example dilute acetic acid, may be used toachieve the removal of the acetone group yielding the diester of FormulaIV.

Before commencing with the next step, the esterification of the freehydroxy groups of the glycerophosphoric acid diester IV, said ester isconverted to a corresponding, heavy metal salt, preferably the bariumsalt. The preparation of this salt serves two purposes. First, a greaterstability is imparted to said ester, facilitating its purification andisolation. Secondly, participation of the acidic hydroxy group of thephosphoric acid portion of the molecule in the esterification step isblocked.

Accordingly, the conversion of the glycerophosphoric acid diester IV tothe corresponding diacyl derivative of Formula V is readily achieved byacylation of a heavy metal salt, for example, the barium salt, of theglycerophosphoric acid diester of Formula IV by conventional methods,such as, for example, the use of an acylating agent, such as, anappropriate acid chloride, in the presence of pyridine. When employingthe conventional method of using an acid chloride in the presence ofpyridine it is an advantage to use dimethylformamide as a solvent forthe reaction, since a homogeneous reaction medium, and consequentlysuperior yields are obtained. When this step is used in the processdirected to the preparation of the compounds of this invention ofgeneral Formula I in which R and R are the same acyl group, an amountmore than two equivalents, preferably five equivalents, of the acylatingagent are employed. On the other hand, for the process directed towardthe compounds of general Formula I in which R and R are different acylgroups, between 0.5 and 1.2 equivalents, preferably 0.8-1.0 equivalent,of the appropriate acylating agent, is first allowed to react with theheavy metal salt of the glycerophosphoric acid diester of Formula IVaccording to the conventional acylating methods, described above, toyield the corresponding derivative possessing an acylated primaryhydroxy group. Subsequent treatment of the latter derivative, also asthe heavy metal salt, for example, the barium salt, with an excess,preferably three equivalents of a different acylating agent, chosenaccordingly, and using the same acylating conditions, affords the diacylderivative of Formula V in which R and R represent different acylgroups.

The preferred acylating agents for the above reactions are thecorresponding acid chlorides, prepared from the appropriate acids,according to the method used by W. Stofi'el and H. D. Pruss, J. LipidRes, 8, 196 (1967) for the preparation of octadeca-6,9,12-trienoylchloride. Such acids are octadeca-6,9,l2-trienoic acid, described by J.M. Osbond and J. C. Wickers, Chem. and 1nd, 1287 (1959);octadeca-9,12,15-trienoic acid, described by S. S. Nigam and B. C. L.Weedon, Chem. and Ind., (1955); octadeca-6,9,12,15-tetraenoic acid,described by M. Matic, Biochem. J., 68, 692 (1958); eicosa-8,ll,l4-trienoic acid, described by W. Stoffel, Ann. Chem, 673, 26(1964); and eicosa-5,8,l1,14-tetraenoic acid, described by J. M. Osbondand J. C. Wickers, cited above.

Finally, the protective group for example, the phthalyl group of thediacyl derivatives of Formula V, is removed by conventional treatmentwith hydrazine hydrate (see for example, E. Schrtider and H. Liibke,cited above) to yield the desired phosphatidylalkanolamine derivativesof this invention.

Alternatively, the compounds of this invention may be synthesized by aprocess illustrated by the following formulae:

in which R R R n and Z are as defined above.

The starting material of Formula II, described above, is condensed inthe presence of an organic base, preferably pyridine, withdi-(2,2,2-trichloroethy1)phosphorochloridate (VI) described by F.Eckstein and K. H. Scheit, Angew. Chem. Internat. edit., 6, 362 (1967),to yield the phosphoric acid triester acetonide of Formula VII. Thetriester acetonide VII is converted to the triester of Formula VIII bymild hydrolyzing methods, such as described above for the conversion ofthe compounds of Formula III to the compounds of Formula IV. Thetriester VIII is then treated with appropriate acylating agents,described above, to yield the diacyl phosphoric acid triester of FormulaIX. The latter compound on treatment with zinc in acetic acid affordsthe corresponding diacyl phosphoric acid monoester of Formula X, whichmay be condensed, for example, in the presence of an organic base,preferably pyridine, and trichloroacetonitrile, with the appropriate 2-or 3-hydroxy-alkylphthalimide, described above, to yield the penultimateintermediate in this process, the phosphatidyl derivative V. Thisphosphatidyl derviative is also the penultimate intermediate for thepreceding process and its conversion to the compounds of this inventionof formula is described above.

Again alternatively, the compounds of this invention may be synthesizedby a process illustrated by the following formulae:

in which R R R and n are as defined above and Z represents a primaryamino group coupled with a tertbutyloxycarbonyl amine-protecting group.

The starting material of Formula II, described above, is condensed inthe presence of an organic base, preferably pyridine with(2,2,2-trichloroethoxy)carbonyl chloride of Formula XI, described by T.B. Windholz and D. B. R. Johnston, Tetrahedron Letters, 2555 (1967), toyield the carbonic acid diester acetonide of Formula XII. The acetonegroup of the acetonide XII is removed to yield the carbonic acid diesterof Formula XIII by mild hydrolyzing methods, such as described above forthe conversion of the compounds of Formula III to the compounds ofFormula IV. The diester XIII is then treated with appropriate acylatingagents, described above, to yield the diacyl carbonic acid diester ofFormula XIV. The latter compound, upon treatment with zinc in aceticacid, readily yields the glycerol diacylate of Formula XV which may becondensed with phosphorus oxychloride in the presence of an Organicbase, preferably quinoline, followed by treatment with an appropriatehydroxyalkylcarbarnic acid tert-butyl ester, described below, to affordthe compounds of. Formula V in which R and R are as defined above and Zrepresents a primary amino group coupled with a tertbutyloxycarbonylgroup.

The appropriate hydroxyalkylcarbamic acid tert-butyl esters preferred inthe above reaction are 2-hydroxyethylcarbamic acid tert-butyl ester,described by F. I. M. Daemen et al., Rec. Trav. Chim., 82, 487 (1963),3-hydroxypropylcarbamic acid tertbutyl ester and 3-hydroxyb'utylcarbamicacid tertbutyl ester. The latter three esters may be readily preparedaccording to the procedure of F. J. M. Daemen et al., cited above, usedfor the preparation of the former ester. When employing this procedurefor this purpose, an equivalent amount of S-amino-l-propanol, describedby L. Henry, Chem. Ber., 33, 3169 (1900), l-amino-Z-propanol, describedby P. A. Levene and J. Scheidegger, J. Biol. Chem, '60, 172 (1924) and4- amino-2-butanol, described by R. Robinson and H. Suginome, citedabove, is used instead of ethanolamine to obtain the correspondinghydroxyalkylcarbamic acid tertbutyl ester, respectively.

The compounds of Formula V in which R and R are as defined above and Zrepresents a primary amino group coupled with a tert-butyloxycarbonylgroup are readily converted to the phosphatidylalkanolamine derivativesof 6 Formula I by conventional methods such as those described by E.Schrtider and K. Liibke, cited above, see page 39; the use of hydrogenchloride in ether solution is a preferred conventional method.

The following examples will illustrate this invention.

EXAMPLE 1 To a stirred solution of 4.6 ml. of freshly distilledphosphorous oxychloride in 25 ml. of dry methylene chloride at 15 C., isadded during min. a solution of 1,2-isopropylidene-sn-glycerol (II, 6.6g.) and 7 ml. of distilled quinoline in ml. of dry methylene chloride.The reaction mixture is kept at 35 C. for 90 min. The temperature of thereaction mixture is again lowered to 10 C. and over a period of 60 min.a solution of 9.5 g. of N-(Z-hydroxyethyl)phthalimide and 16.1 ml. ofdry pyridine in ml. dry methylene chloride is added. The reactionmixture is left at room temperature for 18 hours. Water (1.25 ml.) isadded to the reaction mixture with continuous stirring and the solventis removed under reduced pressure at a temperature not exceeding 40 C.The oily residue is triturated successively with three 125 ml. portionsof petroleum ether and three 125 ml. of dry ether and then exhaustivelyextracted with six 125 ml. portions of benzene. The combined benzeneextract is evaporated to dryness under reduced pressure at EEO-35 C.affording 1,2 isopropylidene sn glycero-3-phosphoryl-N-(2-hydroxyethyl)phthalimide (III; R =H, n=1 and Z=phthalimido),

1775 and 1740 cm.- as a glassy residue. This residue is dissolved in 250ml. of distilled water and allowed to stand at room temperature for 4hours, then freed from insoluble materials by extraction with ether. Tothe clear aqueous solution, 8.75 g. of barium carbonate is added slowlywith continuous stirring. After one hour the mixture is filtered and thefiltrate centrifuged to remove colloidal particles. The clearsupernatant is evaporated under reduced pressure at 3540 C. to yield aglassy solid. This solid is readily soluble in Water, methanol anddimethylformamide and insoluble in ether, benzene and chloroform. It ischaracterized as the barium salt of sn-glycero- 3 phosphoryl N(2-hydroxyethyl)-phthalimide by its analysis: Calculated for C H N O PBa (percent): N, 3.39 and P, 7.50. Found (percent): N, 3.40 and P, 7.67.

In the same manner, but using an equivalent amount ofN-(3-hydroxypropyl)-, N-(2 hydroxypropyl)-, orN-(3-hydroxybutyl)phthalimide instead of N-(Z-hydroxyethyl)phthalimide,the glycerophosphoryl-N-hydroxyalkylphthalimides, snglycero-3-phosphoryl-N-(3-hydroxypropyl)-, -N-(2-hydroxypropyl)-, and-N-(3-hydroxybutyl)- phthalimides and their corresponding barium saltsare obtained, respectively.

In the same manner, but using an equivalent amount ofrac-1,2-isopropylidineglycerol instead of1,2-isopropylidene-sn-glycerol, and the appropriate N-(2- orN-(3-hydroxyalkyl)phthalimide, described above, the correspondingracemic mixtures of sn-glycero-3-phosphoryl-N-(2-hydroxyethyl)-,-N-(3-hydroxypropyl)-, -N-(2-hydroxypropyl)-, and-N-(3-hydroxybutyl)phthalimides, and their corresponding barium salts,are obtained.

EXAMPLE 2 To a solution of the thoroughly dried barium salt of theglycerophosphoryl-N-hydroxyalkylphthalimide, sn-

7. glycero-S-phosphoryl-N-(2-hydroxyethyl)phthalimide IV; R =H, n-=1 andZ phthalimido, 2.8 g., prepared as described in Example 1, in 20 ml. ofdimethylformamide, is added 5.0 g. of the freshly prepared acyl chlorideoctadeca-9,12,15-trienoyl chloride, and 2.7 ml. of dry pyridine. Thesolution is stored under nitrogen at 50 for 48 hours. It is poured intoice and 2 N sulfuric acid. The separated oil is extracted with ether.The ether extract is Washed with cold Water and dried over sodiumsulfate. Removal of solvent gives an oil. The oil is subjected tochromatography using a silicic acid column (40 x 4 cm.). Elution withpetroleum ether, benzene mixtures (1:1 and 1:3) and benzene givescompounds showing a negative phosphate test. Elution .of the column with5% methanol in benzene affords the desiredphosphatidyl-N-hydroxyal-kylphthalimide, 1,2 di(octadeca-9,12,15-trienoyl)-snglycero-3-phosphoryl-N-(2-hydroxyethyl)phthalimide(V; R =H, n=1 and Z=phthalirnido),

.ggq 3400-3250 1775, 1740 and 1720 cmr In the same manner, but using theacid chlorides, octadeca-6,9,12-trienoyl chloride,octadeca-6,9,12,15-tetraenoyl chloride, eicosa-8,11,l4-trienoyl chlorideor eicosa- 5,8,11,14-tetraenoyl chloride instead of octadeca-9,12,l5-trienoyl chloride, the phosphatidyl-N-hydroxyalkylphthalimides,1,2 di(octadeca-6,12,15-trienoyl)-, 1,2-di-(octa- 6,9,12,15-tetraenoyl)-,di-(eicosa-8,11,14-trienoyl)- and di (eicosa 5,8,11,14tetraenoyl-sn-glycero-3-phosphory1)-N-(Z-hydroxyethyl)-phthalimides, areobtained, respectively.

In the same manner, but using the acid chlorides,octadeca-6,9,12-trienoyl chloride, octadeca-9,12,15-trienoyl chloride,octadeca-6,9,12,lS-tetraenoyl chloride, eicosa- 8,11,14-trienoylchloride or eicsa-5,8,11,14-tetraenoyl chloride and the appropriateglycerophosphoryl-N-hydroxyalkylphthalimides, prepared asdescribed inExample 1, the corresponding, phosphatidyl-N-hydroxyalkylphthalimides,1,2-di-(octadeca-6,9,l2-trienoyl), 1,2-di- (octadeca 9,12,15 trienoyl),1,2-di-(octadeca-6,9,12,15- tetraenoyl),1,2-di-(eicosa-8,11,14-trienoyl), 1,2-di-(eicosa- 5,8,11,14-tetraenoyl)esters of glycerophosphoryl-N-hydroxyalkylphthalimides,sn-glycero-3-phosphoryl-N-(3-hydr0xypropyl)-, -N-(2-hydroxypropyl)-,-N-(3-hydroxybutyl)-phthalimides, are obtained.

In a like manner, the manipulative procedure described in this examplemay be employed using an appropriate acid chloride, described above, andglycerophosphoryl- N-hydroxyalkylphthalimide, prepared as described inExample l, in a molar ratio of 0.8:1, followed by a repetition of saidmanipulative procedure using a three molar excess of a different acidchloride, described above, relative to the amount of saidglycerophosphoryl-N-hydroxyalkylphthalimide, to yield the mixeddiacylated phosphatidyl-N-hydroxyalkylphthalimides,1-(octadeca-6,9,12-trienoyl) 2 (octadeca-9,12,15-trienoyl)-,-2-(octadeca-6,9, 12,l-tetraenoyl)-, -2-(eicosa-8,11,14-trien0yl)-,-2-(eicosa-5,8,l1,14-tetraenoyl)-; 1-(octadeca-9,12,15-trienoyl)-2-(octadeca-6,9,12-trienoyl)-, -2-(octadeca 6,9,12,15-tetraenoyl)-, 2-(eicosa-8,1 1,14-trienoyl) 2- (eicosa-5,8,1 1, l4-tetraenoyl)-; 1(octadeca 6,9,12,15 tetraenoyl)-2- (octadeca-6,9,12-trienoyl)-,-2-(octadeca 9,12,15 trienoyl)-, -2-(eic0sa-8,11,l4-trienoyl)-,-2-(eicosa-5,8,l1,14- tetraenoyl)-;1-(eic0sa-8,11,14trienoyl)-2-(octadeca-6,9, 12-trienoy1)-2-(octadeca-9,12,15-trienoyl)-, -2- (octadeca-6,9,12,15-tetraenoyl)-,'-2-(eicosa-5,8,11,14 tetraenoyl)-;1-(eicosa-5,8,11,14-tetraenoyl)-2-(octadeca-6,9,12- trienoyl)-,-2-(octadeca-9,12,15-trienoyl)-, 2-(octadeca6, 9,12,15-tetraenoyl)2-(eicosa-8,l1,14-trienoyl)-sn-glycero-3-phosphoryl-N- (Z-hydroxyethyl-N- 3-hydroxypropyl)-, -N-(2-hydroxypropyl)- and -N-(3-hydroxybutyl)phthalimides.

In the same manner, but using the correspondingracg1ycerophosph0ry1-N-hydroxyalkylphthalimide, prepared as described inExample 1 instead of thesn-glycerophosphoryl-N-hydroxyalkylphthalimides, employed above in thisexample, the corresponding rac-phosphatidyl-N-hydroxyal'kylphthalimidesdescribed in this example, are obtained.

EXAMPLE 3 To a solution of 2.5 g. of thesn-phosphatidyl-N-hydroxyalkylphthalimide,1,2-di-(octadeca-9,12,15-trienoyl)- sn-glycero 3 phosphoryl N (2hydroxyethyl)phthalimide, prepared as described in Example 2, in 60 ml.of dry ethanol at 0, 1.25 ml. of a 12.5% hydrazine hydrate solution isadded and kept at said temperature for 30 min. Another 1.8 m1. of thesame hydrazine solution is added and the mixture is boiled undernitrogen for 2 hours. The solvent is removed under reduced pressure andthe residue extracted with ether. The ether extract is mixed with 5 ml.of methanol and 5 ml. of Water and the mixture shaken With 2 g. of Dowex50 (I-I resin for one hour. The mixture is filtered and the filtrate isconcentrated to dryness under reduced pressure. The residue is subjectedto chromatography on a column of silicic acid (4 x 30 cm.). The columnis exhaustively eluted, first With benzene-chloroform (1:2) and thenwith chloroform-methanol (9:1). The chloroform-methanol eluates areconcentrated to yield the sn-phosphatidylethanolamine, 1,2-di-(octadeca-9,12,15 trienoyl) sn-glycero-3-phosphorylethanolamine,

1735, 1650 and 1245 cmr In the same manner, but using equivalent amountsof the other sn-phosphatidyl-N-hydroxyalkylphthalirnides, prepared asdescribed in Example 2, instead of 1,2-di- (octadeca 9,12,15 trienoyl)sn glycero 3 phosphoryl-N-(2-hydroxyethyl phthalamide, the correspondingphosphatidylethanolamines, 1,2 di (octadeca- 6,9,12 trienoyl)-, 1,2 di(octadeca 6,9,12,15-tetraenoyl)-, 1,2 di (eicosa 8,11,14 trienoyl)- and1,2- di (eicosa 5,8,11,14 tetraenoyl) sn glycero 3-phosphorylethanolamines, are obtained. Similarly, the corresponding snphosphatidylethanolamine derivatives, 1,2 di (octadeca 6,9,12 trienoyl),1,2 di (octadeca 9,12,15 trienoyl), 1,2 di (octadeca 6,9,12,15-tetraenoyl), 1,2 di (eicosa 8,11,14 trienoyl), 1,2- di (eicosa 5,8,11,14tetraenoyl) esters of sn glycero- 3 phosphoryl (3 hydroxypropylamine),-(2 hydroxypropylamine), and -(3 hydroxybutylamine), are obtained.Similarly, the corresponding mixed acylated snphosphatidylethanolaminederivatives, 1 (octadeca- 6,9,12 trienoyl) 2 (octadeca 9,12,15trienoyl)-, -2 (octadeca 6,9,12,15 tetraenoyl)-, -2 (eicosa- 8,11,14trienoyl)-, -2 (eicosa 5,8,11,14 tetraenoyl)-; 1 (octadeca 9,12,15trienoyl) 2 (octadeca 6,9,12- trienoyl)-, -2 (octadeca 6,9,12,15tetraenoyl)-, -2- (eicosa 8,11,14 trienoyl)-, -2 (eicosa 5,8,11,14-tetraenoyl)-; 1 (octadeca 6,9,12,15 tetraenoyl) 2- (octadeca 6,9,12,15trienoyl)-, -2 (octadeca 9,12,15- trienoyl)-, -2 (eicosa 8,11,14trienoyl)-, -2 (eicosa- 5,8,11,14 tetraenoyl)-; 1 (eicosa 8,11,14trienoyl)- 2 (octadeca 6,9,12 trienoyl)-, -2 (octadeca 9,12,15-trienoyl)-, -2 (octadeca 6,9,12,15 tetraenoyl)-, -2- eicosa 5,8,11,14tetraenoyl)-; 1 (eicosa 5,8,11,14- tetraenoyl) 2 (octadeca 6,9,12trienoyl) -2 (octadeca 9,12,15 trienoy1)-, 2 (octadeca 6,9,12,15-tetraenoyl)-, -2 (eicosa 8,11,14 trienoyl) sn glycero- 3 phosphoryl 2ethanolamines, -3 hydroxypropylamines, -2 hydroxypropylamines and -3hydroxybutylamines, are obtained.

In the same manner, but using the correspondingracphosphatidyl-N-hydroxyalkylphthalimides, prepared as described inExample 2 instead of the sn-phosphatidyl- N-hydroxyalkylphthalimidesemployed above in this example, the correspondingrac-phosphatidylethanolamine derivatives are obtained.

9 EXAMPLE 4 To a stirred solution of 45.1 g. ofdi(2,2,2-trichloroethyl)phosphorochloridate (VI) in 300 ml. of drypyridine at C., 12.6 g. of 1,2 isopropylidine sn glycerol (II) in 50 ml.of pyridine is added dropwise over a period of 30 minutes. The reactionmixture is kept at 0 C. for 12 hours and then concentrated under reducedpressure. The residue is dissolved in chloroform and the resultantsolution washed with dilute sodium bicarbonate. Concentration of thechloroform solution yields 1,2-isopropylidine sn glycero 3 phosphoricacid di (2,2,2- trichloroethyl)-ester (VII),

6 9 1.35 (s, 3 protons) 1.45 (s, 3 protons) and 4.75 (s, 4 protons).

In the same manner, but using an equivalent amount of rac 1,2isopropylidene glycerol instead of 1,2 isopropylidene sn glycerol, rac1,2 isopylidene glycerol- 3 phosphoric acid di (2,2,2trichloroethyl)ester is obtained.

EXAMPLE 5 A solution of 5.0 g. of 1,2 isopropylidene sn glycero 3phosphoric acid di (2,2,2 trichloroethyl)- ester (VII), prepared asdescribed in Example 4, in 250 ml. of 80% methanol is passed through acolumn of Dowex 50 (H+). The column is rinsed with one liter of 80%methanol. The combined elfiuents are evaporated at reduced pressure at35-40 C., yielding sn glycero 3- phosphoric acid di (2,2,2trichloroethyl) ester (VIII),

CHOlz max.

3600 cm.- (broad), 6

EXAMPLE 6 By using the manipulative procedures and the appropriate acidchlorides, described in Example 2, but substituting an equivalent amountof snor rac-glycero- 3-phosphroic acid di-(2,2,2-trichloroethyl)-ester,prepared as described in Example 5 in place of the glycerophosphoryl Nhydroxylalkylphthalimide, followed by treatment with an excess of zincdust in 80% aqueous acetic acid for one hour at room temperature toremove the 2,2,2-trichloroethyl ester groups the corresponding snorracphosphatidic acid derivatives, 1,2 di (octadeca 6,9,12- trienoyl)-,1,2 di (octadeca 9,12,15 trienoyl)-, 1,2- di (octadeca 6,9,12,15tetraenoyl)-, 1,2 di (eicosa- 8,11,14 trienoyl)-, 1,2 di (eicosa5,8,11,14 tetraenoyl)-; 1 (octadeca 6,9,12 trienoyl) 2 (octadeca-9,12,15 trienoyl)-, -2 (octadeca 6,9,12,15 tetraenoyl)-, -2 (eicosa8,11,14 trienoyl)-, -2 (eicosa- 5,8,1l,14 tetraenoyl)-; 1 (octadeca9,12,15 trienoyl) 2 (octadeca 6,9,12 trienoyl)-, -2 (octadeca- 6,9,12,15tetraenoyl)-, -2 (eicosa 8,11,14 trienoyl)-, -2 (eicosa 5,8,11,14tetraenoyl)-; 1 (octadeca- 6,9,12,15 tetraenoyl) 2 (octadeca 6,9,12trienoyl)-, -2 (octadeca 9,12,15 trienoyl)-, -2 (eicosa 8,11,14-trienoyl)-, -2 (eicosa 5,8,11,14 tetraenoyl)-; l-(eicosa- 8,11,14trienoyl) 2 (octadeca 6,9,12 trienoyl)-, -2 (octadeca 9,12,15trienoyl)-, -2 (octadeca 6,9,12,15 tetraenoyl)-, -2 (eicosa 5,8,11,14tetraenoyl)-; 1 (eicosa 5,8,11,14 tetraenoyl) 2 (octadeca 6,9,12trienoyl)-, -2 (octadeca 9,12,15 trienoyl)-, 2 (octadeca 6,9,12,15tetraenoyl)-, -2- (eicosa 8,11,14 trienoyl)-, glycero 3 phosphoricacids, are obtained.

The 1,2 di (octadeca 9,12,15 trienoyl) snglycero 3 phosphoric acid (X; Rand R =CH (CH CH=CH)3(CH COO) has 173 59 1750 cm.- (broad) EXAMPLE 7 Asolution of 7.0 g. of the phosphatidic acid derivative 1,2 di-(octadeca9,12,15 trienoyl)-sn-glycero-3-phosphoric acid, prepared as described inExample 5, 1.8 g. of the hydroxyalkylphthalimide, N-(2hydroxyethyl)phtha1- imide, and 5.00 g. of trichloroacetonitrile,described by W. Steinkopf, Chem. Ber., 41, 2540 (1908), in 40 ml. ofpyridine is heated at -100 C. for four hours. The reaction mixture isdiluted with water (300 ml.) adjusted to pH 6 with concentratedhydrochloric acid, mixed with ice, and extracted with chloroform. Thechloroform extract is dried over sodium sulfate, filtered and evaporatedto dryness. The residual oil is subjected to chromatography using asilicic acid column (40X 8 cm.). After elution of the column withpetroleum ether, petroleum ether-benzene (1:1) and benzene, elution with5% methanol in benzene affords 1,2 di (octadeca 9,12,15trienoyl)-sn-glycero- 3-phosphoric acid,

vCHCh 3400-3250 In the same manner, but using the appropriate snorrac-phosphatidic acid derivative, prepared as described in Example 6,and the appropriate 2- or 3-hydroxyalkylphthalimide, described above,the corresponding snor rac-phosphatidyl-N hydroxyalkylphthalimides,described in Example 2, are also obtained.

EXAMPLE 8 A solution of 1.0 g. of (2,2,2-trichloroethoxy)carbonylchloride (X1) in 5 ml. of dry ether is added slowly to a vigorouslystirred ice-cold solution of 500 mg. of 1,2-isopropylidine-sn-glycerol(II) and 1 ml. of pyridine in 5 ml. of dry ether. After stirring at roomtemperature for one hour, the reaction mixture is diluted with ether.The resultant precipitate of pyridine hydrochloride is removed byfiltration. The filtrate is washed successively with cold 2N-hydrochloric acid and water, dried over sodium sulfate, filtered andevaporated to dryness. The oily product, 1,2 isopropylidine-sn-glycero 3carbonic acid (2,2,2- trichloroethyl) -ester (XII),

H l D .35 1750 cm. ag f 1.35 (s, 3 protons), 1.45 (s, 3 protons), 4.75(s, 2 protons) is not purified further but used for the next stepdescribed in Example 9.

In the same manner, but using an equivalent amount of rac 1,2isopropylidine-glycerol instead of 1,2 isopropylidine-sn-glycerol, rac1,2 isopropylidine-glycero-3- carbonic acid (2,2,2-trichloroethyl)esteris obtained.

EXAMPLE 9 To a solution of 1.1 g. of 1,2 isopropylidine-sn-glycero- 3carbonic acid (2,2,2 trichloroethyl)-ester (XII), prepared as describedin Example 8, in 20 ml. of methanol, 0.1 ml. of concentratedhydrochloric acid is added. The resultant solution is kept at roomtemperature for one hour and then evaporated to dryness under reducedpressure. After drying in high vacuum for 24 hours over sodiumhydroxide, the oily residue, sn-glycero-S-carbonic acid (2,2,2trichloroethyl)-ester (XIII),

11,9 ,39 3500 and 1750 emf- 52 35 4.75 (s, 2 protons) By using themanipulative procedures and the appropriate acid chlorides, described inExample 2, but substituting an equivalent amount of snor rac-glycero 3carbonic acid (2,2,2 trichloroethyl)-ester, prepared as described inExample 9 in place of the glycerophosphoryl-N-hydroxyalkylphthalimide,followed by treatment with an excess of zinc dust in glacial acetic acidfor one hour at room temperature to remove the 2,2,2-trichlorethyl estergroup the corresponding snor rac-glycerol diacylate derivatives, 1,2di-(octadeca-6,9,12 trienoyl)-, 1,2 di- (octadeca 9,12,15 trienoyl)-,1,2 di-(octadeca 6,9, 12,15 tetraenoyl)-, 1,2 di-(eiscosa 8,11,14trienoyl)-, 1,2 di-(eicosa 5,8,11,14 tetraenoyl)-; 1 (octadeca- 6,9,12trienoyl) 2 (octadeca 9,12,15 trienoy1)-, -2- (octadeca 6,9,12,15tetraenoyl)-, -2 (eicosa 8,11,14- trienoyl)-,-2-(eicosa-5,8,11,14-tetraenoyl)-; l-(octadeca- 9,12,15 trienoyl) 2(octadeca 6,9,12 trienoy1)-, -2- (octadeca 6,9,12,15 tetraenoyl)-,-2-(eicosa 8,11,14- trienoyl)-, -2-(eicosa 5,8,11,14 tetraenoyl)-;l-(octadeca 6,9,12,15 tetraenoyl) -2 (octadeca 6,9,12-trienoyl)-,-2-(octadeca 9,12,15 trienoyl)-, -2-(eicosa 8, 11,14 trienoyl)-,-2(eicosa 5,8,11,14-tetraenoyl); 1- (eicosa 8,11,14 trienoyl) 2(octadeca 6,9,10 trienoyl)-, -2-(octadeca-9,12,15-trienoyl)-,-2-(octadeca-6,9, 12,15-tetraenoyl)-, -2-(eicosa-5,8,11,14 tetraenoyl)-;1- (eicosa 5,8,11,14 tetranoyl) 2 (octadeca 6,9,12- trienoyl)-,-2-(octadeca-9,12,15-trienoyl)-, 2-(octadeca-6, 9,12,15-tetraenoyl)-,-2-(eicosa 8,11,14,,- trienoyl), glycerols are obtained.

The 1,2 di-(octadeca 9,12,15 trienoyl)-sn-glycerol :(XV); :R and R =CH(CH -CH=CH) (CH COO has V359 3400 and 1745 cm.-

EXAMPLE 11 To a solution of 0.46 g. of freshly distilled phosphorousoxychloride in ml. of dry methylene chloride at 0 0,, is added during 30min. a solution of 1,2 di-(octadeca- 9,12,15 trienoyl)-sn-glycerol,prepared as described in Example 10, and 0.43 g. of dry quinoline in 10ml. of dry methylene dichloride. The reaction mixture is kept at roomtemperature for one hour. The temperature of the reaction mixture isagain reduced to 0 C. and a solution of 0.48 g. of2-hydroxyethylcarbamic acid tert-butyl ester and 0.8 g. of pyridine in10 ml. of methylene chloride is added over a period of 30 min. Afterstanding at room temperature for 2 hours, 0.05 ml. of water is added andthe reaction mixture is stirred for one hour, diluted 'with 200 ml. ofmethylene chloride and washed successively with cold 2 N hydrochloricacid and water, dried over sodium sulfate, filters and concentratedunder reduced pressure to yield an oil. The oil is purified bychromatography on silicic acid. Elution with 5% methanol in benzeneaffords the phosphatidyl derivative,1,2-di-(octadeca-9,12,lS-trienoyl)-sn-glycero 3phosphoryl-N-(tert-butyloxycarbonyl)ethanolamine (V); R and R =CH (CH CHCH 3 (CH COO R =H, n =1 and Z=NHCOOC(CH ,CHOla max.

3500-3400, 1745 and 1690 cmr This derivative is dissolved in ml. of dryether and treated with a stream of dry hydrogen chloride at 0 C. for twohours. The solvent is then removed by under reduced pressure at 30 C.The oily residue is subjected to chromatography on g. of silicic acid.Elution with 510% methanol in chloroform yields the desiredphosphatidylalkanolamine, 1,2-di-(octadeca 9,12,15 trienoyl)-sn-glycero3 phosphorylethanolamine,

ase 3500-3100, 1735, 1650 and 1245 cmr identical with the productobtained in Example 3.

In the same manner, but using the appropriate snor rac-glyceroldiacylate described in Example 10 instead of 1,2 di-(octadeca 9,12,15trienoyl)-sn-glycerol together with the appropriate hydroxyalkylcarbamicacid tert-butyl ester, described above, instead of2-hydroxyethylcarbamic acid tert-butyl ester, the remainingphosphatidylalkanolamines described in Example 3 are obtained.

I claim:

1. 1,2 di-(octadeca 9,12,15 trienoyl)-sn-glycero-3-phosphorylethanolamine.

References Cited UNITED STATES PATENTS 2,629,662 2/1953 Julian et al260-403 2,864,848 12/ 1958 McArthur 260-403 3,189,628 6/1965 Knight etal. 260-403 ELBERT L. ROBERTS, Primary Examiner US. Cl. X.R.

